Compositions and methods for treating hypophosphatasia

ABSTRACT

The present invention provides compositions and methods for use in enzyme replacement therapy. The inventors disclose a method of producing membrane bound enzymes in an active soluble form by eliminating the glycosylphosphatidylinositol (GPI) membrane anchor. In particular the inventors disclose a soluble active form of the membrane bound enzyme TNSALP which they produced by deleting the GPI anchor single peptide sequence. They have further shown that this composition is useful for treatment of hypophosphatasia. The inventors also disclose oligo acid amino acid variants thereof which specifically target bone tissue.

PARENT CASE TEXT

This application is a divisional application of U.S. patent applicationSer. No. 11/484,870 filed Jul. 11, 2006 pending and claims benefit ofpriority to U.S. Provisional Patent Application No. 60/725,563, filedOct. 11, 2005. All documents above are incorporated herein in theirentirety by reference.

SEQUENCE LISTING

A paper copy of the sequence listing and a computer readable form of thesame sequence listing are appended below and herein incorporated byreference. The information recorded in computer readable form isidentical to the written sequence listing, according to 37 C.F.R. 1.821(f).

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to compositions and methods of enzymereplacement therapy (ERT). More specifically, the invention is directedto compositions and methods for treatment of enzyme deficient diseasesuch as hypophosphatasia using a genetically modified polynucleotide toproduce in an active secretory form of alkaline phosphatase.

2. Description of the Related Art

Alkaline phosphatase (ALP) is a ubiquitous plasma membrane-bound enzyme.Hypophosphatasia is an inherited metabolic disorder of defective bonemineralization caused by deficiency of a form of ALP know astissue-nonspecific alkaline phosphatase (TNSALP). Clinical severity isremarkably variable, ranging from death in utero to merely prematureloss of dentition in adult life [1, 2]. Despite the presence of TNSALPin bone, kidney, liver, and adrenal tissue in healthy individuals,clinical manifestations in patients with hypophosphatasia are limited todefective skeletal mineralization that manifests as rickets in infantsand children and osteomalacia in adults [2]. In the most pernicious formof hypophosphatasia, the perinatal lethal variant, profound skeletalhypomineralization results in caput membranaceum with shortened anddeformed limbs noted. Some affected neonates survive for several days orweeks. They often succumb to respiratory failure brought on by pulmonaryhypoplasia and structural failure of the weakened skeleton fromdemineralization [3].

Osteoblasts modulate the composition of the bone matrix, where theydeposit mineral in the form of hydroxyapatite. Specialized buds from theosteoblasts' plasma membrane are called matrix vesicles (MVs). Theinitiation of matrix calcification by osteoblasts and chondrocytesappears to be mediated by release of MVs, which serve as a shelteredenvironment for hydroxyapatite crystal formation [4-7]. MVs are alkalinephosphatase enriched, extracellular, membrane-invested bodies. InsideMVs the first crystals of hydroxyapatite bone mineral are generated.TNSALP hydrolyzes inorganic pyrophosphate (PP_(i)) to monophosphate(inorganic phosphate; P_(i)), which is important for growth of thehydroxyapatite crystal [4, 5, 8-10]. Thus ALP functions as an inorganicpyrophosphatase (PP_(i)-ase) [14, 15]. PP_(i) itself impairs the growthof hydroxyapatite crystals as an inhibitor of mineralization [8, 11-13].Insufficient TNSALP activity fails to hydrolyze PP_(i) and the resultingbuild-up of unhydrolyzed PP_(i) in the perivesicular matrix inhibits theproliferation of pre-formed hydroxyapatite crystals beyond theprotective confines of MV membranes.

The level of plasma PP_(i) increases in hypophosphatasia [16-18]. Evenin the absence of TNSALP, the other phosphatases (AMPase and inorganicpyrophosphatase) can hydrolyze PP_(i), supplying P_(i) for incorporationinto initial mineral within MVs [19] but still be insufficient to removeexcess PP_(i) at the perimeter of MVs. Thus, despite TNSALP deficiency,initial mineral could form within MVs, while its propagation intoperivesicular matrix would be inhibited by a local build-up ofPP_(i [)20, 21]. These findings suggest PP_(i) as a plausible candidateas an inhibitor of mineralization and as a primary factor that causesclinical manifestations of hypophosphatasia.

Enzyme replacement therapy (ERT) has proven effective in preventing orreversing lysosomal storage in patients and animal models with lysosomalstorage diseases (LSDs) [22-28]. Tremendous progress in the developmentof ERT has been made in the last three decades. Cellular uptake ofenzyme from the blood following intravenous administration requiresspecific oligosaccharides on the enzyme itself corresponding tooligosaccharide receptors on the target cells. Examples include thebinding of high-mannose oligosaccharides of the enzyme to the mannosereceptor (MR) and binding of phosphorylated high-mannoseoligosaccharides of the enzyme to the cation-independent mannose6-phosphate receptor (M6PR). Thus, LSDs have been considered potentiallyamenable to therapy with exogenously supplied enzymes.

The cell-specific delivery system was also designed to enhance theclinical effectiveness of ERT. In the case of Gaucher disease, deliveryof the enzyme to the affected cells was achieved by modifying theN-linked carbohydrate on the enzyme. This exposed core mannose residues[29, 30], enabling the enzyme to bind to the MR, which is highlyabundant on cells of the reticuloendothelial system [31, 32]. Thesefindings led to clinical management of Gaucher disease by ERT [22]. Over3,500 patients have been treated with dramatic clinical results [33].

However, hypophosphatasia caused by a deficiency of TNSALP seems to be adifficult disorder treated by ERT because TNSALP is a membrane-boundenzyme and is believed to require attachment at the cell surface to befunctional. In fact, the results of multiple intravenous infusions ofplasma ALP or purified liver ALP in patients with hypophosphatasia havebeen disappointing [34-38]. Administration of exogenous pyridoxal HCldelayed the onset of epileptic attacks and increased the life span ofTNSALP−/− mice. Although the oldest survivor was 22 days old, all thehomozygotes, however, died near weaning time, irrespective of theirtreatment regime [39].

The inventors have genetically engineered a Chinese Hamster Ovarian(CHO) cell line to produce a C-terminus-anchorless TNSALP enzyme, insecreted form, [40] and showed clinical effectiveness of ERT onhypophosphatasia mice. These results indicate that theC-terminus-anchorless membrane enzyme possesses the characteristicsnecessary for use in ERT where the membrane-binding form is ineffective.Deletion of the C-terminus membrane anchor will be applicable to othermembrane-binding proteins whose deficiency leads to other humandisorders including but not limited to paroxysmal nocturnalhaemoglobinuria (PNH).

Targeted therapies have the advantage of reducing adverse effects onnon-target organs as well as reducing the minimum effective systemicdose. Recently, Kasugai et al [41] has demonstrated that a small peptideconsisting of a stretch of acidic amino acids (L-Aspartic acid orL-Glutamic acid) was selectively delivered to and retained in bone aftera systemic administration. Furthermore, a small molecule, an estrogen,conjugated with an acidic-oligopeptide, has been selectively targeted tobone, leading to dramatic improvement of the bone mineral density inovariectomized mice with no or few adverse effects to liver and uterus[42]. However, whether such a bone-targeting system with an acidicoligopeptide could be applied to a large molecule such as an enzyme in amanner such that the enzyme is functional and efficiently producedremains unsolved.

The inventors have sought to address the issue of enzyme replacementtherapy using membrane bound enzymes genetically modified to besynthesized in an active secretory form. In particular the inventorshave applied this method to TNSALP as a treatment for hypophosphatasia.This method of releasing membrane bound enzymes in a functional formwill offer new avenues for therapeutic strategies to combat disease ofenzyme deficiency.

SUMMARY OF THE INVENTION

The inventors have made the surprising discovery that removal of thenucleotide sequence encoding the C-terminus glycosylphosphatidylinositol(GPI) anchoring signal peptide of a membrane bound enzyme and expressingthat nucleotide sequence in a host cell, will result in the synthesisand extracellular release of an active enzyme in a soluble form.Furthermore, a membrane bound enzyme such as tissue-nonspecific alkalinephosphatase (TNSALP) in an anchorless form is useful in enzymereplacement therapy for treatment of hypophosphatasia.

Hypophosphatasia, caused by deficient activity of TNSALP results indefective bone mineralization. Plasma infusions of TNSALP have notachieved clinical improvement. No definitive treatment is presentlyavailable. Enzyme replacement therapy for hypophosphatasia was notthought to be feasible since TNSALP exists as a membrane-bound enzymeand functions physiologically when the enzyme is present at the cellmembrane. A tissue TNSALP knock-out mouse provides a model of infantilehypophosphatasia displaying impaired bone mineralization, epilepticseizures, apnoea, and abnormal apoptosis in the thymus, abnormal lumbarnerve roots, and postnatal death before the weaning.

To investigate the clinical effectiveness of ERT for hypophosphatasia,the inventors deleted the C-terminus of TNSALP cDNA encoding the GPIanchoring signal peptide sequence and transfected the modifiednucleotide into the Chinese hamster ovary (CHO) cell line. The resultwas a secreted form of anchorless recombinant human TNSALP (anchorlessrhTNSALP) produced by CHO cells, which was subsequently purified andcharacterized in vitro.

An in vivo study was carried out, which utilized weekly infusions ofanchorless rhTNSALP into TNSALP knockout mice. In vitro mineralizationassays with anchorless rhTNSALP in the presence of high concentrationsof pyrophosphate provided evidence of bone mineralization with bonemarrow from a hypophosphatasia patient. Administration of the purifiedanchorless rhTNSALP enzyme into TNSALP knockout mice increased life spanand increased body weight, showing that the treated mice livedapproximately 4 and 7 times longer compared to the untreated mice.Treated mice had no epileptic seizures until at least 3 months old.

These results show the C-terminus anchorless rhTNSALP functionsbioactively in vivo and that is a good candidate for ERT forhypophosphatasia. This invention can be applied to other diseasesdeficient in membrane-bound proteins.

Targeted therapies are often advantageous because they can reduceoverall total effective dose and in turn adverse consequences topatients. To this purpose the inventors tagged anchorless rhTNSALPenzymes with an acidic oligopeptide, of six or eight residues ofL-Aspartic acid, to provide high affinity binding to hydroxyapatitewhich is abundant in bone. The inventors characterized the biochemicalproperties of the purified tagged enzymes in comparison with theuntagged enzyme to evaluate the feasibility of bone-directionaldelivery. CHO cell lines were established producing the taggedanchorless rhTNSALP enzymes as a secreted form. It was found thatspecific activities of the purified enzymes tagged with the acidicoligopeptide were almost the same as the untagged enzyme. In vitroaffinity measurements indicated that the poly-aspartic acid taggedenzymes had an approximately 10-fold higher affinity to hydroxyapatitethan the untagged TNSALP enzyme. Lectin affinity chromatography showedlittle difference among the tagged and untagged enzymes in carbohydratestructure except the tagged enzymes had fewer sialic acid residues.Biodistribution pattern analysis by infusion of the fluorescence-labeledenzymes into mice showed that the amount of the tagged enzymes retainedin bone was 4-fold higher than that of the untagged enzyme at 6 hourspost-infusion. The tagged enzymes were retained at higher levelscontinuously up to one week.

These results indicate that the enzymes tagged with an acidicoligopeptide are delivered more specifically to bone and possess a highaffinity for hydroxyapatite, suggesting the potential use of the taggedenzymes in targeted ERT for hypophosphatasia.

Therefore, an object of this invention is a method of modifying amembrane bound protein by eliminating the GPI anchor such the protein isnot bound to the cell membrane and may exist extracellularly in asoluble active form.

In another embodiment, the object of this invention is a TNSALP,modified so that it does not comprise a GPI anchor, and that thisanchorless TNSALP is not bound to the cell membrane and may existextracellularly in a soluble active form such that it may be usedtherapeutically in enzyme replacement therapy for ALP deficient diseasessuch as hypophosphatasia.

In another embodiment, the object of this invention is a TNSALP,modified such that the TNSALP does not comprise a GPI anchor and thisanchorless TNSALP is not bound to the cell membrane and may existextracellularly in a soluble active form, and further comprises anacidic oligopeptide sequence, such as poly-aspartic acid, providing ahigh affinity for bone tissue so that it may be used therapeutically inERT for ALP deficient diseases such as hypophosphatasia.

In another embodiment, the invention is drawn to a method ofmanufacturing an ALP ERT factor, comprising the steps of a) deleting theGPI anchor signal peptide encoding sequence form a nucleotide, b)transfecting a cell with said modified nucleotide, c) culturing thecell, and d) purifying the ALP ERT factor form the culture media.

In yet another embodiment the invention is drawn to a method of treatinga patient with hypophosphatasia using ALP ERT factors.

It is envisioned that the instant ALP ERT factors (supra) may beadministered to patients in vivo, in a pharmaceutically acceptableformulation as a therapy for the treatment of hypophosphatasia, orencoded a nucleotide sequence to be expressed in cells within a patientto supply the aforementioned factors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Construct of anchorless TNSALP. The glycosylphosphatidylinositol(GPI) anchoring signal peptide sequence of TNSALP (SEQ ID NO: 2) wasdeleted from (A) the full-length of TNSALP cDNA to produce (B) cDNAencoding the secreted form of the enzyme.

FIG. 2. SDS-PAGE of ALP ERT factors from condition medium. The purifiedenzymes (0.2 μg) were subjected to SDS-PAGE under reducing condition andstained with silver. A Single band appeared in all the three enzymes.The molecular mass of the untagged anchorless rhTNSALP (lane 1) wasapproximately 80 kDa, while those of CD6- and CD8-TNSALP were larger(lanes 2 and 3, respectively).

FIG. 3. Concentration-dependent binding curves of anchorless TNSALP andtagged anchorless TNSALP to hydroxyapatite. Purified enzymes were mixedwith a hydroxyapatite suspension at a final concentration of 1.0, 2.5,5.0, and 10.0 μg/ml. The mixture was mixed at 37° C. for 1 h, andcentrifuged at 14,000×rpm for 10 min to separate bound and unboundenzymes. To determine the amount of the unbound enzyme, the enzymeactivity in supernatant was measured. The amount of bound enzyme wasdetermined by measuring both total and unbound enzymes. Affinity forhydroxyapatite for oligo Aspartic acid tagged enzymes was 10-fold higherthan that for the untagged enzyme. Also binding to hydroxyapatite wasseen at lower concentrations of Aspartic acid tagged enzyme.

FIG. 4. ConA affinity chromatography of three ALP ERT factors.Anchorless rhTNSALP (A), CD6-TNSALP (B), and CD8-TNSALP (C) were appliedto a ConA affinity column. After washing the column, two fractions wereeluted by two different concentrations, 0.01 M (arrow; a) and 0.5 M(arrow; b) of αMM. There was no difference in the elution profile amongthe three enzymes.

FIG. 5. WGA affinity chromatography of ALP ERT factors. ALP ERT factorsbefore (A-C) and after (D-F) the neuraminidase digestion were applied tothe WGA affinity chromatography. The anchorless rhTNSALP (A and D),CD6-TNSALP (B and E), and CD8-TNSALP (C and F) enzymes were applied tothe WGA column. After washing the column, two fractions were eluted bythe two different concentrations, 0.1 M (arrow; a) and 0.5 M (arrow; b)of GlcNAc.

FIG. 6. SDS-PAGE of ALP ERT factors before and after neuraminidasedigestion. The enzymes (0.3 μg) were subjected to SDS-PAGE underreducing condition and stained with silver. A single band was observedat all the lanes. After the treatment with neuraminidase, the molecularmass of the three enzymes decreased in a similar proportion.

FIG. 7. Biodistribution of fluorescence-conjugated ALP ERT factors tobone. Fluorescence-labeled ALP ERT factors, (A) anchorless rhTNSALP, (B)CD6-TNSALP, and (C) CD8-TNSALP, were infused to mice from tail vein atthe dose of 1 mg/kg of body weight. At the indicated time points 6, 24,72, and 168 hours (1, 2, 3, and 4 respectively), the legs were dissectedand sectioned. The sections of legs were observed under a fluorescentmicroscopy to evaluate the enzyme distribution at the epiphyseal region.ALP ERT factors were distributed to the mineralized region (m), but notto the, growth plate (gp).

FIG. 8. Relative area of fluorescence around growth plate after a singleinfusion of fluorescence-ALP ERT factors. The average of the relativeareas of fluorescence from three fields of the fluorescent images atepiphyseal region was quantitated.

FIG. 9. In vitro mineralization experiment with anchorless rhTNSALPenzyme. The bone marrow cells derived from a hypophosphatasia patientwere seeded in 12-well plate at a density of 10,000 cells/cm2, anddifferentiated under existing 2.5 mM Pi or 2.5 mM β-glycerophosphate asa phosphate source. The effect on mineralization of anchorless rhTNSALPenzyme was evaluated in the presence of PPi. The calcium deposits werevisualized 12 days after the initiation of differentiation of bonemarrow cells.

FIG. 10. Clinical phenotype of TNSALP (−/−) mouse treated by anchorlessrhTNSALP. The upper mouse is a wild-type from the same littermate whilethe lower mouse is treated with anchorless rhTNSALPfor 6 weeks. Thestature and appearance of treated mouse is nearly the same as thewild-type control mouse.

FIG. 11 Growth curve of mice injected with anchorless rhTNSALP of 5mg/kg. A) Specimen 1, B) Specimen 2. TNSALP (−/−) mouse which receivedenzyme on the day after birth, followed by further weekly injection upto 10 weeks. At 0, 1, 2, 3, 4 weeks, the enzyme was injected byintraperitoneal. After 5 weeks through 10 weeks, enzyme was injectedthrough tail vein weekly (black diamond line). The wild-type littermatesof the treated TNSALP (−/−)(open circles). The untreated TNSALP (−/−).The untreated mice died before the weaning (x-x).

DETAILED DESCRIPTION OF THE INVENTION

In vivo, TNSALP is bound to plasma membranes by a GPI anchor, which isadded after removal of a C-terminus peptide during post-translationalprocessing. TNSALP functions as an ectoenzyme. In this study, theinventors have removed the nucleotide sequence encoding the GPI anchorsignal from human TNSALP cDNA in order to express and secrete ananchorless form of TNSALP into the culture medium of overexpressingCHO-K1 cells. This study demonstrates that removal of the GPI anchoringsignal peptide sequence from the C-terminus of TNSALP cDNA allows theoverexpressing CHO-K1 cells to produce sufficient amounts of recombinanthuman enzyme in a secreted form, and that this anchorless recombinanthuman TNSALP enzyme is bioactive and able to initialize bonemineralization in bone marrow from hypophosphatasia patients. Inaddition, when anchorless rhTNSALP was infused into the sublethal formof TNSALP (−/−) mouse, it improved clinical features and increased bothlife span and growth, further indicating the feasibility of enzymereplacement therapy for hypophosphatasia.

Hypophosphatasia is a metabolic bone disease that establishes animportant role for alkaline phosphatase (ALP) in skeletalmineralization. Subnormal serum ALP activity (hypophosphatasemia)constitutes the biochemical hallmark and reflects a generalizeddeficiency of activity of the tissue-nonspecific (liver/bone/kidney) ALPisoenzyme (TNSALP). Activities of the three tissue-specific ALPisoenzymes in humans—intestinal, placental, and germ-cell(placental-like) ALP—are not diminished. TNSALP is a zincmetalloglycoprotein that is catalytically active as a multimer ofidentical subunits. It is bound to plasma membranes by GPI linkage.

Hypophosphatasia is characterized clinically by defective skeletalmineralization that manifests as rickets in infants and children andosteomalacia in adults. Clinical expressivity is, however, extremelyvariable. Stillbirth can occur from in utero onset in the perinatal(“lethal”) form, which is apparent in newborns and associated with themost severe skeletal hypomineralization and deformity. The infantileform presents as a developmental disorder by age 6 months. It may causecraniosynostosis and nephrocalcinosis from hypercalcemia andhypercalciuria and is often fatal. Premature loss of deciduous teeth andrickets are the cardinal clinical features of childhoodhypophosphatasia. Adult hypophosphatasia typically results in recurrentmetatarsal stress fractures and pseudofractures in long bones andoccasionally produces arthritis from calcium pyrophosphate dihydrate(CPPD) and perhaps calcium phosphate crystal deposition.Odontohypophosphatasia refers to especially mildly affected individualswho have dental, but no skeletal, manifestations.

Three phosphocompounds [phosphoethanolamine (PEA), PPi, and pyridoxal5′-phosphate (PLP)] accumulate endogenously in hypophosphatasia and areinferred to be natural substrates for TNSALP. A variety of evidenceshows that PLP, a cofactor form of vitamin B6, collects extracellularly;intracellular levels of PLP are normal. This observation explains theabsence of symptoms of deficiency or toxicity of vitamin B6 andindicates that TNSALP functions as an ectoenzyme. Extracellularaccumulation of PPi, which at low concentrations promotes calciumphosphate deposition but at high concentrations acts as an inhibitor ofhydroxyapatite crystal growth, appears to account for the associatedCPPD deposition and perhaps calcific periarthritis, as well as thedefective mineralization of bones and teeth. There is no establishedmedical treatment. Enzyme replacement by IV infusion of ALP from varioustissue sources has generally not been of significant clinical benefit[34-38]. Therefore, it has long been thought that since TNSALP is amembrane-bound protein, via GPI linkage, TNSALP needs to be attached tothe membrane to provide a physiological function.

In this study the inventors have established a newly designed ERT forhypophosphatasia with C-terminus anchorless recombinant human TNSALP andhave shown clinical effectiveness with the TNSALP (−/−) mouse model.This strategy is applicable to other GPI-anchored proteins whosedysfunction leads to the human disorders such as paroxysmal nocturnalhaemoglobinuria (PNH) and prion diseases.

Bone Targeted Anchorless rhTNSALP

The development of selective drug delivery to bone will enhance theclinical effectiveness of bioactive enzymes used in ERT. To thispurpose, the inventors have invented an acidic-oligopeptide-taggedbone-directional anchorless rhTNSALPs for use in ERT, and havecharacterized these enzymes for their bone-targeting properties. Theinventors tagged the anchorless rhTNSALP enzymes with an acidicoligopeptide (a six or eight stretch of L-Aspartic acid), to provide ahigh affinity for hydroxyapatite, which is abundant in bone. Theinventors characterized the biochemical properties of the purifiedtagged enzymes in comparison with the untagged enzyme to evaluate thefeasibility of the bone-directional delivery.

CHO cell lines producing tagged (six or eight residues of L-Asparticacid) and untagged anchorless rhTNSALP enzymes were established. Thespecific activity of purified enzymes tagged with the acidicoligopeptides was almost identical with the untagged enzyme. In vitroaffinity assays showed that the tagged anchorless rhTNSALPs had a10-fold higher affinity for hydroxyapatite than the untagged anchorlessrhTNSALP. Lectin affinity chromatography showed little difference incarbohydrate structure among the tagged and untagged enzymes except forfewer sialic acid residues on the tagged enzymes. The examination ofbiodistribution patterns after a single infusion of fluorescence-labeledALP ERT factors into mice showed that the amount of tagged enzymesretained in bone were 4-fold higher than that of the untagged enzyme at6 hours post-infusion. The tagged enzymes were retained continuously ata higher level up to one week.

These results show that ALP ERT factors tagged with an acidicoligopeptide are characterized with a more specific affinity binding tothe hydroxyapatite, suggesting the potential use of the tagged enzymesfor ERT on hypophosphatasia.

Therefore, the invention is drawn to (1) a method of producing ananchorless membrane bound protein in a soluble active form, by deletingthe GPI anchoring signal peptide, (2) composition and manufacture of ananchorless human recombinant TNSALP (anchorless rhTNSALP) for treatmentof hypophosphatasia by deleting the GPI anchoring signal peptide nucleicacid sequence from cDNA and transfecting a host cell for high yieldexpression and release of the enzyme, (3) a method and composition foran acidic oligopeptide tagged variant of anchorless rhTNSALP fortargeted delivery to bone, and (4) methods of using anchorless rhTNSALPand oligopeptide tagged variants of anchorless rhTNSALP to treathypophosphatasia in a patient.

The term “anchorless recombinant human TNSALP” or “anchorless rhTNSALP”refers to the tissue non-specific alkaline phosphatase (TNSALP) whichhas been modified by deletion of the GPI anchor. The term “TNSALP”generally referees to tissue non-specific alkaline phosphatase. As usedin FIGS. 1, 3, 6, and 8 as well as the provisional application to whichthis application claims priority, TNSALP or rhTNSALP, where it isapplicably described, is equivalent to, anchorless human recombinantTNSALP or anchorless rhTNSALP.

The terms “CD6-TNSALP” and “CD8-TNSALP” or “CD6” and “CD8” refer to theanchorless recombinant human TNSALP or anchorless rhTNSALP which havebeen tagged with 6 or 8 L-aspartic acid residues attached at thecarboxyl terminus respectively. The term “tagged” or “oligopeptidetagged” means the act of adding to, in this case, referring to theadding of six or eight aspartic acids residues to anchorless rhTNSALPthrough genetic engineering or other chemical means.

The term “ALP” refers to the family of alkaline phosphatase enzymesgenerally.

The term “ERT” refers to enzyme replacement therapy for treatment ofdisease. A disease caused by enzyme deficiency treated throughreplacement of the deficient enzyme. As used here it refers toreplacement of the deficient enzyme, by way of explanation but not oflimitation, inter venous infusion or administration of a corrective geneor cell containing a corrective gene to produce the deficient enzyme ina patient.

The term “ALP ERT factors” refers generally to alkaline phosphataseenzymes useful in enzyme replacement therapy. More specifically thisterm is meant to include all compositions of anchorless rhTNSALP,CD6-TNSALP and CD8-TNSALP disclosed herein.

The term “GPI anchor” is meant to refer to glycosylphosphatidylinositolattached at or near the C-terminus of a membrane bound protein, therebybinding the membrane bound protein to the membrane via its lipidphilicaffinity with the membrane.

The term “GPI anchor signal peptide” is meant to refer to the C-terminusamino acid sequence recognized during post-translational processing as asingle for adding GPI and thereby anchoring the protein.

The term “GPI anchor single peptide sequence” refers to a nucleotidesequence encoding the GPI anchor signal peptide.

The term “active” means a functional state of a molecule where itperforms as it would in vivo, including reactions the enzymes is know tofacilitate or binding or blocking functions receptors may be know topossess. Active also includes any pro-active state, pro-enzymes whichnormally exist in a precursor from; that is not capable of carrying outtheir known function until activated by another factor or co-factor.

Sequence identity or percent identity is intended to mean the percentageof same residues between two sequences. The two reference sequences usedare the entire peptide sequence of human tissue non-specific alkalinephosphatase precursor (residues 1-524), or the GPI anchor single peptideof human tissue non-specific alkaline phosphatase precursor (residues506-524). In all sequence comparisons, the two sequences being comparedare aligned using the Clustal method (Higgins et al, Cabios 8:189-191,1992) of multiple sequence alignment in the Lasergene biocomputingsoftware (DNASTAR, INC, Madison, Wis.). In this method, multiplealignments are carried out in a progressive manner, in which larger andlarger alignment groups are assembled using similarity scores calculatedfrom a series of pairwise alignments. Optimal sequence alignments areobtained by finding the maximum alignment score, which is the average ofall scores between the separate residues in the alignment, determinedfrom a residue weight table representing the probability of a givenamino acid change occurring in two related proteins over a givenevolutionary interval. Penalties for opening and lengthening gaps in thealignment contribute to the score. The default parameters used with thisprogram are as follows: gap penalty for multiple alignment=10; gaplength penalty for multiple alignment=10; k-tuple value in pairwisealignment=1; gap penalty in pairwise alignment=3; window value inpairwise alignment=5; diagonals saved in pairwise alignment=5. Theresidue weight table used for the alignment program is PAM250 (Dayhoffet al., in Atlas of Protein Sequence and Structure, Dayhoff, Ed., NBRF,Washington, Vol. 5, suppl. 3, p. 345, 1978).

TABLE 1 Percent Identity of ALPs. Species Accession number PercentIdentity Human tissue non- NP_000469 100 specific alkaline phosphataseprecursor Rhesus tissue non- XP_001109717 97 specific alkalinephosphatase Rat tissue-nonspecific NP_037191 90 alkaline phosphatase Dogtissue non-specific AAF64516 89 alkaline phosphatase Pig alkalinephosphatase AAN64273 88 Shown are calculations of percent identity forcomparison of alkaline phosphatase from various mammalian speciesrelative to human tissue non-specific alkaline phosphatase precursor.

TABLE 2 Percent Identity of GPI anchor single peptide. Species Accessionnumber Percent Identity Human tissue non- NP_000469 100 specificalkaline phosphatase precursor (residues 506-524) Rhesus tissue non-XP_001109717 84 specific alkaline phosphatase residues (634 652) Pigalkaline phosphatase AAN64273 75 (residues 237-253) Dog tissuenon-specific AAF64516 68 alkaline phosphatase (residues 487-502) Rattissue-nonspecific NP_037191 NP_599169 68 alkaline phosphatase (residues509-524) Shown are calculations of identity for comparisons of GPIanchor single peptide sequences from various mammalian species relativeto the GPI binding signal peptide of human tissue non-specific alkalinephosphatase precursor.

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims, which follow the examples.

Example 1 Preparation and Biochemical Characterization of Enzymes

The GPI anchoring signal peptide was removed from the carboxyl-terminalof the human TNSALP to release the enzyme in the media of CHO-K1 cells.This was accomplished by deleting the GPI anchoring signal sequence fromfull length TNSALP cDNA (FIG. 1). The resultant anchorless rhTNSALPenzyme (>95%) was mainly secreted to culture medium in a transientexpression study (data is not shown). Acidic oligopeptide-tagged enzymes(CD6-TNSALP and CD8-TNSALP), which also lack the GPI anchoring signalpeptide, were secreted in to the culture medium as well. Constructs forthe CD6- to CD8-TNSALP cDNA were made and transfected into CHO-1 cellsfor transient expression. Cells stably expressed and secreted activeTNSALP enzymes into the medium in linear fashion for 12 h. Howeverexpression of enzyme plateaued after 12 hours. The inventor's previouswork with oligopeptide-tagged enzymes showed that increasing the numberof Aspartic acid residues beyond eight caused a substantial reduction ofenzyme activity secreted into culture media in the transient expression(data not shown). The inventors chose the 6 and 8 aspartic acid taggedenzymes (CD6-TNSALP and CD8-TNSALP) for further evaluation as theirexperience had shown that these molecules will exhibit superiorexpression characteristics.

The purification of these enzymes was performed by a two-step columnchromatography method, using DEAE-Sepharose and Sephacryl S-400, assummarized in Table 3. The overall purification yields of anchorlessrhTNSALP, CD6-TNSALP, and CD8-TNSALP were 32%, 62%, and 56% of the totalenzymes in the culture media, respectively, and the specific activitiesof each enzyme were 2744, 2411, and 2374 units/mg, respectively. Thelower purification yield of anchorless rhTNSALP than those of the taggedenzymes was apparently due to a broader peak eluted from the DEAEcolumn.

TABLE 3 Purification of rhTNSALP and acidic oligopeptide-tagged TNSALPfrom condition medium Protein concen- Total Total Specific trationprotein activity activity Purifi- (mg/l) (mg) (units/mg) (units/mg)cation Yield rhTNSALP Crude 5.26 115 3003 26.1 1 100 media DEAE 18.30.66 1555 2354 90 52 column Sephacryl 15.4 0.35 973 2744 105 32 S-400-HRColumn CD6-TNSALP Crude 6.27 127 3022 23.9 1 100 media DEAE 32.3 1.012073 2043 86 69 column Sephacryl 22.1 0.77 1862 2711 101 62 S-400-HRColumn CD8-TNSALP Crude 3.85 184 3065 16.6 1 100 media DEAE 29.1 1.002028 2035 123 66 column Sephacryl 22.4 0.72 1702 2374 143 56 S-400-HRColumn

When the purified anchorless rhTNSALP was subjected to SDS-PAGE underreducing conditions, a single band with approximately 80 kDa ofmolecular mass was detected (FIG. 2). An increase of molecular massassociated with an additional acidic oligopeptide was observed in CD6-and CD8-TNSALP.

There was little difference among anchorless rhTNSALP, CD6-TNSALP, andCD8-TNSALP in Michaelis constant (KM), as defined by the pNPP substratewith double-reciprocal plots (0.37, 0.39, and 0.37 mM, respectively), orin chemical inhibition by L-phenylalanine (10 mM; 83%, 86%, and 86% ofremaining enzyme activity, respectively) and L-homoarginine (10 mM; 12%,13%, and 12% of remaining enzyme activity, respectively).

Example 2 Characteristics of Poly-Aspartic Acid—Tagged AnchorlessrhTNSALP Affinity for Hydroxyapatite

A remarkable difference between the tagged and untagged enzymes wasobserved in their affinity to hydroxyapatite. Affinity to hydroxyapatitefor the tagged enzymes was 10-fold higher than that for the untaggedenzyme and the binding to hydroxyapatite was seen even at lowconcentration of the tagged enzyme (FIG. 3). The binding parameters,K_(b) and B_(max), are shown in Table 4. The values of K_(b) and B_(max)of the tagged enzymes were 10- and 3-fold, respectively, higher thanthose of the untagged enzyme. Although no significant difference wasobserved between CD6- and CD8-TNSALP.

TABLE 4 Binding parameters of three enzymes to hydroxyapatite. KbB_(max) (ug/100 ug (ug⁻¹ml) hydroxyapatite) rhTNSALP  1.7 ± 1.0 0.5 ±0.2 CD6-TNSALP 36.7 ± 7.9 1.6 ± 0.3 CD8-TNSALP 44.6 ± 4.6 1.9 ± 0.7 Eachvalue represents the mean ± S.D. of 3 experiments. K_(b) bindingconstant and B_(max) maximum binding rates were determined formdouble-reciprocal plots.

Elution Profiles of Enzymes by Lectin Affinity Chromatography

Three enzymes, rhTNSALP, CD6-, and CD8-TNSALP, were subjected to ConAaffinity chromatography. (FIG. 4). ConA affinity chromatographyindicated there was little unbound enzyme, whereas weakly-bound andstrongly-bound enzymes were detected. Overall the elution profiles ofthese enzymes did not differ when two different concentrations ofcompetitive sugars were added. Since ConA has a high reactivity to themannosyl residues, the inventors concluded that these enzymes did notdiffer with respect to mannosyl residue composition. In contrast, theWGA elution profiles between the tagged and untagged enzymes wereremarkably different in the ratio of strongly-bound enzyme andweakly-bound enzyme (FIG. 5A-C). Table 5 shows the percentages of therelative enzyme activity of three fractions on the WGA column.Approximately 30% of the tagged enzymes were weakly bound and 70% wasstrongly bound to the WGA column, while 66% of the untagged enzyme wasweakly bound and 34% was strongly bound to the WGA column. The contentof the weakly-bound enzyme was larger in the order ofrhTNSALP>CD6-TNSALP>CD8-TNSALP.

TABLE 5 Percentage of Unbound, Weakly bound, and Strongly boundfractions obtained by each ConA and WGA column Percent of relativeactivities ConA WGA WGA + Neuraminidase rhTNSALP CD6 CD8 rhTNSALP CD6CD8 rhTNSALP CD6 CD8 Unbound 3 4 2 0 3 1 0 2 4 Weakly 59 60 59 66 32 239 11 4 Bound Strongly 38 36 39 33 65 76 91 86 92 Bound

To estimate the content of the sialic acid residues of the enzyme, wetreated three enzymes with neuraminidase thereby removing the sialicacid residues from the enzymes. After the treatment with neuraminidase,the molecular masses of three enzymes decreased in a similar proportion(FIG. 6). The elution profile of the untagged enzyme on the WGA columnchanged after the neuraminidase digestion. The earlier fractionaccounting for the weakly-bound enzyme shifted to the later fraction forthe highly-bound enzyme (FIG. 5D). On the other hand, the elutionprofiles of the tagged enzymes on the WGA column slightly changed withneuraminidase digestion (FIGS. 5E and 5F), since the tagged enzymesoriginally included a less amount of weakly-bound enzyme.

Biodistribution of Fluorescence-Labeled Enzymes

To evaluate the pharmacokinetic tissue distribution pattern of theseenzymes, the fluorescence-labeled enzymes were prepared by the Alexadye. The efficiencies of labeling in each of three enzymes wereapproximately 10 mol/mol of protein as dye content. FIG. 7 shows thehistological pictures of biodistribution of three enzymes at theepiphyseal region at 6, 24, 72, and 168 h after a single intravenousinfusion. FIG. 8 shows the average of the relative area of fluorescence.Three enzymes were distributed to the mineralized region, but not to thegrowth plate. At 6 h, the relative areas of fluorescence at the taggedenzymes were four-fold larger than the area at the untagged enzyme.Moreover, the fluorescence-labeled tagged enzymes retained until 168 hwith two- to three-fold larger amount than the untagged enzyme. Theseresults were consistent with the result of the in vitro hydroxyapatiteaffinity experiment. In liver, relatively high amount of enzymedistribution was observed compared to other tissues (data not shown).The distribution was widespread throughout the liver includinghepatocytes and sinus-lining cells. The distribution patterns in liverwere comparable among three enzymes. In other tissues including brain,lung, heart, spleen, and kidney, no significant difference was observedamong three enzymes as well (data not shown).

Overall, the above results showed no biochemical and pharmacokineticdifference between two tagged enzymes.

Example 3 Effect of Anchorless rhTNSALP on Mineralization in thePresence of PP_(i)in Primary Bone Marrow Cell Culture

In human bone marrow cells derived from a hypophosphatasia patient,mineralization never occurred in the absence of TNSALP even whenβ-glycerophosphate was added. The addition of one of the enzyme resultedin marked recovery of mineralization (FIG. 9). In contrast,mineralization was observed when P_(i) was used in the medium instead ofβ-glycerophosphate even in the absence of any enzyme. The presence ofany of the enzymes did not provide any additive effect for themineralization. These findings indicate that the anchorless rhTNSALPenzyme played a biological role in the mineralization process byproviding free P_(i) released during the hydrolysis ofβ-glycerophosphate. We added PP_(i) an inhibitor of mineralization, tosee whether the anchorless rhTNSALP enzyme hydrolyze PP_(i) to restorethe mineralization. PP_(i) itself completely inhibited themineralization even in the presence of P_(i). The addition of the enzymerestored the mineralization level to PP_(i)-free control culture.

Example 4 Enzyme Replacement Therapy with Anchorless rhTNSALP

The TNSALP gene knock-out mouse strains as models for hypophosphatasiahad <1% of wild-type plasma TNSALP activity. These TNSALP−/− mice weregrowth impaired, develop epileptic seizures and apnea, and died beforeweaning as described previously [39, 47, 48]. Postnatal growth ofTNSALP−/− mice treated with anchorless rhTNSALP at 5 mg/kg of bodyweight and their littermate controls are shown in FIG. 10. The averagelife span of untreated TNSALP−/− mice without anchorless rhTNSALP enzymeadministration was 10 days [39, 47, 48]. In treated mice, injected withanchorless rhTNSALP, no epileptic seizures appeared until at least 2months old, in addition the mice lived approximately 4 and 7 times aslong. Growth curves of TNSALP−/− mice and littermate controls withouttreatment are shown in FIG. 11 for comparison. One mouse treated with IPinfusion for 4 weeks did not grow well (FIG. 11A). However after IVinfusion began, the mouse increased its body weight substantially. Asecond mouse treated with IV infusion at birth grew well at subnormallevels (FIG. 11B). Both of these mice exhibited no abnormal activity andseizures.

Overall, ERT with the C-terminus anchorless rhTNSALP enzyme showedclinical effectiveness on TNSALP−/− mice.

Materials and Methods

Production of human recombinant acidic oligopeptide-tagged and untaggedTNSALPs (GenBank: NM_(—)000478.2)—The GPI anchoring signal peptidesequence of TNSALP(5′-CTTGCTGCAGGCCCCCTGCTGCTCGCTCTGGCCCTCTACCCCCTGAGCGTCCTGT TC-3′:c.1516C to c.1572C: Leu506 to Phe524) (SEQ ID NO: 2) was deleted fromthe full-length of TNSALP cDNA to produce the enzymes as a secretedform. To produce acidic oligopeptide-tagged TNSALP, a stretch of six oreight of L-Asp (six L-Asp, 5′-GACGATGACGACGATGAT-3′ (SEQ ID NO: 3):eight L-Asp, 5′-GATGATGATGATGATGATGACGAC-3′(SEQ ID NO: 4)) wasintroduced additionally at the C-terminus after c.1515C of Ser505 (CD6-or CD8-TNSALP, respectively) mediating a linker(5′-ACCGGTGAAGCAGAGGCC-3′ (SEQ ID NO: 5)), followed by a terminationcodon. The three enzymes used for the further experiments were named asanchorless rhTNSALP (human TNSALP anchorless at the C-terminal),CD6-TNSALP (human TNSALP anchorless at the C-terminal tagged with astretch of six L-Asp), and CD8-TNSALP (human TNSALP anchorless at theC-terminal tagged with a stretch of eight L-Asp), respectively.

For the preparation of the first strand cDNA, reverse transcriptasereaction was performed by using total RNA isolated from healthy humanperipheral blood. To amplify rhTNSALP, CD6-TNSALP, and CD8-TNSALP cDNA,PCR reactions were carried out with the following primers: TNSALP,forward 5′-GAATTCACCCACGTCGATTGCATCTCTGGGCTCCAG-3′ (SEQ ID NO: 6) andreverse 5′-ctcgagTCAGCTGCCTGCCGAGCTGGCAGGAGCAC-3′(SEQ ID NO: 7):CD6-TNSALP, forward 5′-GAATTCACCCACGTCGATTGCATCTCTGGGCTCCAG-3′ (SEQ IDNO: 8) and reverse5′-tcaatcatcgtcgtcatcgtcggcctctgcttcaccggtGCTGCCTGCCGAGCTGGCAGGAGCACAGTG-3′(SEQID NO: 9): CD8-TNSALP, forward5′-GAATTCACCCACGTCGATTGCATCTCTGGGCTCCAG-3′ (SEQ ID NO: 10) and reverse5′-tcagtcgtcatcatcatcatcatcatcggcctctgcttcaccggtGCTGCCTGCCGAGCTGGCAGGAGCACAGTG-3′(SEQ ID NO: 11). The nucleotide sequences compatible with six oreight of L-Asp were added to the reverse primers used here. Theamplified cDNA were cloned and sequenced. The cDNA were then transferredinto EcoRI cloning sites of mammalian expression vector pCXN, kindlyprovided by Miyazaki J., Osaka University, Suita, Japan (40).

The anchorless rhTNSALP, CD6-TNSALP, and CD8-TNSALP cDNAs subcloned inpCXN were then transfected into Chinese hamster ovary (CHO-K1) cellswith lipofectamine according to manufacture's instruction (Invitrogen).Selection of colonies was carried out in growth medium with Dulbecco'sModified Eagle Medium supplemented with 15% fetal bovine serum (FBS),plus 600 μg/ml G418 (Sigma-Aldrich) for 10-12 days. Individual cloneswere picked, grown to confluency, and analyzed for enzyme expression bymeasuring secreted enzyme activity in the medium as described below. Thehighest-producing clone was grown in collection medium with Ex-Cell™ 325PF CHO Protein-free medium (JRH Biosciences) and 15% FBS. When the cellsreached confluency, the cells were rinsed with PBS and fed withcollection media without FBS to collect enzyme for purification.

Measurement of Alkaline Phosphatase Activity

A 50 μl of volume of sample was combined with 250 μl of 10 mMp-nitrophenyl phosphate (pNPP) (Sigma-Aldrich, MO) as a substrate in 1 Mdiethanolamine, pH 9.8, containing 1 mM magnesium chloride and 0.02 mMzinc chloride, and incubated at 37° C. The time-dependent increase inabsorbance at 405 nm (reflecting p-nitrophenolate production) wasmeasured on a plate spectrophotometer (EL800, Bio-Tek Instrument, Inc.,VT). One unit of activity was defined as the quantity of enzyme thatcatalyzed the hydrolysis of 1 μmol substrate in 1 min.

Enzyme Purification

The anchorless rhTNSALP enzyme was purified by a two-step columnprocedure. Tris buffer was 25 mM Tris-HCl, pH 8.0, containing 0.1 mMmagnesium chloride and 0.01 mM zinc chloride. Unless stated otherwise,all steps were performed at 4° C.

Step 1. The medium containing enzyme was filtered through a 0.2 μmfilter, and then dialyzed against Tris buffer using Amicon stirred-cellultrafiltration unit with Millipore ultrafiltration membrane YM-30.

Step 2. The dialyzed medium was applied to a column of DEAE Sepharose(Sigma-Aldrich, MO) equilibrated with Tris buffer. The column was firstwashed with Tris buffer, and then the enzyme was eluted with 0-0.4 MNaCl in a linear gradient.

Step 3. The active eluted fractions were pooled and dialyzed againstTris buffer containing 0.1 M NaCl by using Centricon centrifugal filterdevice with Millipore ultrafiltration YM-10 filter. The dialyzedfractions were then concentrated for step 4.

Step 4. The concentrated enzyme was applied to a column of SephacrylS-400-HR (Sigma-Aldrich, MO) equilibrated with Tris buffer containing0.1 M NaCl. The enzyme was eluted with Tris buffer containing 0.1 MNaCl.

Step 5. The active eluted fractions were pooled and dialyzed againstTris buffer containing 0.1 M NaCl by using Centricon centrifugal filterdevice with Millipore ultrafiltration YM-10 filter. The dialyzedfractions were then concentrated and stored at −80° C. until use.

Polyacrylamide Gel Electrophoresis

Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE) was performed, followed by silver staining [44, 45].

Hydroxyapatite binding assay—Hydroxyapatite beads (Sigma-Aldrich) weresuspended in 25 mM Tris-HCl buffered saline (TBS), pH 7.4, atconcentration of 100 μg/100 μl. The purified enzyme was mixed with thehydroxyapatite suspension at a final concentration of 1.0, 2.5, 5.0, and10.0 μg/ml. The mixture was mixed at 37° C. for 1 h, and centrifuged at14,000×rpm for 10 min to separate unbound enzyme and bound enzyme. Todetermine unbound enzyme, enzyme activity in supernatant was measured,and bound enzyme was determined from the amount of total enzyme andunbound enzyme. Binding constant (K_(b)) and maximal binding rate(B_(max)) were determined from double-reciprocal plots.

Lectin Affinity Chromatography

To evaluate the carbohydrate chain structure of the enzymes, we appliedthe enzymes to lectin affinity chromatography. TBS used here was 10 mMTris-HCl, pH 8.0, supplemented with 0.5 M sodium chloride, 1 mM calciumchloride, 1 mM magnesium chloride, 1 mM manganese chloride and 0.01 mMzinc chloride. The column of the concanavalin A (Canavalia ensiformis,ConA)-sepharose 4B (Sigma-Aldrich) and the wheat germ agglutinin(Triticum vulgaris, WGA)-agarose CL-4B (Fluka) were equilibrated withTBS at a flow rate of 0.2 ml/min. Lectin affinity chromatography wasperformed as described previously [43]. Briefly, the purified enzyme in0.6 ml of TBS was applied to the ConA and WGA columns, and left to standfor 3 h at room temperature. Three fractions were obtained by using twodifferent concentrations, 0.01 M and 0.5 M of α-methyl-D-mannopyranoside(αMM) (Sigma-Aldrich) from ConA column, and 0.1 M and 0.5 M ofN-acetyl-D-glucosamine (GlcNAc) (Sigma-Aldrich) from the WGA column:unbound fraction, weakly-bound fraction, and strongly-bound fraction.

Neuraminidase Digestion

Tagged and non-tagged rhTNSALPs were digested with α(2 3, 6, 8, 9)neuraminidase (Arthrobacter ureafaciens) (Sigma-Aldrich) to clarify thecontent of sialic acids at the carbohydrate chain. Twenty units of eachpurified TNSALP enzyme were exposed to 0.01 unit of neuraminidase in 250mM sodium phosphate, pH 6.0, overnight at room temperature. The digestedenzyme was then analyzed for polyacrylamide gel electrophoresis andlectin affinity chromatography, as described above.

Biodistribution of Alexa-Labeled Enzymes

One mg/ml of purified enzymes were labeled with Alexa Fluor 546 ProteinLabeling Kit following manufacture's instruction (Molecular Probes). TheAlexa-labeled enzyme was injected to B6 mice (6-7 weeks old) from tailvein at a dose of 1 mg/kg of body weight. Mice were sacrificed at 6, 24,72, and 168 h after a single infusion, and multiple tissues includingbrain, lung, heart, liver, spleen, kidney, and leg were dissected. Thetissues were immersion-fixed in 10% neutral buffered formalin, embeddedin paraffin, and sectioned. Tissues were studied by fluorescencemicroscopy for evaluation of enzyme distribution, and the areas offluorescence from three fields of fluorescent images around growth platewere quantitated by using AlphaEaseFC (Alpha Innotech Corp.).

In Vitro Mineralization Assay

To evaluate the level of bioactivity of the anchorless rhTNSALP enzyme,in vitro mineralization experiments were performed using bone marrowcells derived from a hypophosphatasia patient with an infantile form (10month old). The bone marrow cells were seeded into 150×25 mm tissueculture dishes. These cells were allowed to attach without disturbancefor seven days in growth medium consisting of minimum essential mediumalpha (MEMα) supplemented with 10% FBS, 50 units/ml penicillin, and 50μg/ml streptomycin sulfate. The medium was then replaced to fresh growthmedium at 3-day intervals. When the cells reached confluency, they weresubcultured in the 12-well plates at a density of 10,000 cells/cm². Onthe following day, the growth medium was replaced with thedifferentiation medium: with MEMα supplemented with 10% FBS, 50 units/mlpenicillin, 50 μg/ml streptomycin sulfate, 0.3 mM ascorbic acid, and 100nM dexamethasone. The differentiation medium also included 2.5 mM P_(i)or β-glycerophosphate as a phosphate source as well as either anchorlessrhTNSALPat 2.5 or 5.0 units/ml. To further investigate the effect of thethree enzymes on mineralization in the presence of PP_(i), 50 μM PP_(i)was added always with each enzyme to the bone marrow cell culturethroughout the differentiation period. The differentiation medium wasreplaced at 3-day intervals. At 12 days after the initiation of thedifferentiation of the cells, the cells were fixed with 4%paraformaldehyde, followed by staining with Alizarin Red S to detectcalcium phosphate deposits [46].

Long Term ERT with Anchorless rhTNSALP to Evaluate ClinicalEffectiveness.

Long term ERT was performed using the anchorless rhTNSALP enzymedescribed above. The cephalic vein is the preferred injection rout atbirth but is not visible after about 1 week. Intraperitoneal injectionswere administered from 1 to 4 weeks until the tail vain became visible.Three littermates remained untreated. Two mice (Specimens 1 and 2)received treatments of 5 mg/kg of body weight. Specimen received enzymeby cephalic vein injection on the day following birth, followed byweekly intraperitoneal injections at 0, 1, 2, 3, and 4 weeks and tailvein injections from 5 through 10 weeks. Similarly a Specimen 2 receivedenzyme on the day following birth by cephalic vein injection, followedby weekly intraperitoneal injections at 1, 2, and 3, weeks after whichinjection was administered though the tail vein From 4 through 10 weeks.

REFERENCES

The following numbered references are cited throughout this disclosure.These references are herein incorporated by reference. Applicantsreserve the right to challenge the veracity of any statement made inthese references.

-   [1] D. Fraser, Hypophosphatasia. Am. J. Med. 22 (1957) 730-746.-   [2] M. P. Whyte, Hypophosphatasia, in: C. R. Scriver, A. L.    Beaudet, W. S. Sly, D. Valle (Eds), The Metabolic and Molecular    Bases of Inherited Disease, eighth ed., McGraw-Hill, New York, 2001,    pp. 5313-5329.-   [3] M. M. Silver, G. A. Vilos, K. J. Milne, Pulmonary    hypophosphatasia in neonatal hypophosphatasia. Pediatr. Pathol.    8 (1988) 483-493.-   [4] S. Y. Ali, Matrix formation and mineralization in bone,    in: C. C. Whitehead (Ed), Bone biology and skeletal disorders,    Carfax Publishing Co., Abingdon, U.K., 1992, pp. 19-38.-   [5] H. C. Anderson, Molecular biology of matrix vesicles. Clin.    Orthop. Relat. Res. 314 (1995) 266-280.-   [6] A. L. Boskey, B. D. Boyan, Z. Schwartz, Matrix vesicles promote    mineralization in a gelatin gel. Calcif. Tissue Int. 60 (1997)    309-315.-   [7] A. L. Boskey, Amorphous calcium phosphate: the contention of    bone. J. Dent. Res. 76 (1997) 1433-1436.-   [8] H. C. Anderson, Mechanisms of pathologic calcification. Rheum.    Dis. Clin. North Am. 14 (1988) 303-319.-   [9] L. F. Bonewald, Z. Schwartz, L. D. Swain, B. D. Boyan,    Stimulation of matrix vesicle enzyme activity in osteoblast-like    cells by 1,25(OH)2D3 and transforming growth factor beta (TGF beta).    Bone Miner. 17 (1992) 139-144.-   [10] K. N. Fedde, Human osteosarcoma cells spontaneously release    matrix-vesicle-like structures with the capacity to mineralize. Bone    Miner. 17 (1992) 145-151.-   [11] H. Fleisch, R. G. Russell, F. Straumann, Effect of    pyrophosphate on hydroxyapatite and its implications in calcium    homeostasis. Nature 212 (1966) 901-903.-   [12] A. S. de Jong, T. J. Hak, P. van Duijn, The dynamics of calcium    phosphate precipitation studied with a new polyacrylamide steady    state matrix-model: influence of pyrophosphate collagen and    chondroitin sulfate. Connect. Tissue Res. 7 (1980) 73-79.-   [13] J. L. Meyer, Can biological calcification occur in the presence    of pyrophosphate? Arch. Biochem. Biophys. 15 (1984) 1-8.-   [14] D. W. Moss, R. H. Eaton, J. K. Smith, L. G. Whitby, Association    of inorganic-pyrophosphatase activity with human    alkaline-phosphatase preparations. Biochem. J. 102 (1967) 53-57.-   [15] F. A. Leon, L. A. Rezende, P. Ciancaglini, J. M. Pizauro,    Allosteric modulation of pyrophosphatase activity of rat osseous    plate alkaline phosphatase by magnesium ions. Int. J. Biochem. Cell    Biol. 30 (1998) 89-97.-   [16] R. G. Russell, S. Bisaz, A. Donath, D. B. Morgan, H. Fleisch,    Inorganic pyrophosphate in plasma in normal persons and in patients    with hypophosphatasia, osteogenesis imperfecta, and other disorders    of bone. J. Clin. Invest. 50 (1971) 961-965.-   [17] E. Sorensen, H. Flodgaard, Adult hypophosphatasia. Acta. Med.    Scand. 197 (1975) 357-360.-   [18] S. A. Sorensen, H. Flodgaard, E. Sorensen, Serum alkaline    phosphatase, serum pyrophosphatase, phosphorylethanolamine and    inorganic pyrophosphate in plasma and urine. A genetic and clinical    study of hypophosphatasia. Monogr. Hum. Genet. 10 (1978) 66-69.-   [19] H. C. Anderson, Pyrophosphate stimulation of calcium uptake    into cultured embryonic bones. Fine structure of matrix vesicles and    their role in calcification. Dev. Biol. 34 (1973) 211-227.-   [20] H. C. Anderson, H. H. Hsu, D. C. Morris, K. N. Fedde, M. P.    Whyte, Matrix vesicles in osteomalacic hypophosphatasia bone contain    apatite-like mineral crystals. Am. J. Pathol. 151 (1997) 1555-1561.-   [21] H. C. Anderson, J. B. Sipe, L. Hessle, R. Dhanyamraju, E.    Atti, N. P. Camacho, J. L. Millan, Impaired calcification around    matrix vesicles of growth plate and bone in alkaline    phosphatase-deficient mice. Am. J. Pathol. 164 (2004) 841-847.-   [22] N. W. Barton, R. O. Brady, J. M. Dambrosia, A. M. Di    Bisceglie, S. H. Doppelt, S. C.-   Hill, H. J. Mankin, G. J. Murray, R. I. Parker, C. E. Argoff, et al    Replacement therapy for inherited enzyme    deficiency-macrophage-targeted glucocerebrosidase for Gaucher's    disease. N. Engl. J. Med. 324 (1991) 1464-1470.-   [23] M. S. Sands, C. Vogler, J. W. Kyle, J. H. Grubb, B. Levy, N.    Galvin, W. S. Sly, E. H.-   Birkenmeier, Enzyme replacement therapy for murine    mucopolysaccharidosis type VII. J. Clin. Invest. 93 (1994)    2324-2331.-   [24] R. M. Shull, E. D. Kakkis, M. F. McEntee, S. A. Kania, A. J.    Jonas, E. F. Neufeld, Enzyme replacement in a canine model of Hurler    syndrome. Proc. Natl. Acad. Sci. 91 (1994) 12937-12941.-   [25] A. C. Crawley, D. A. Brooks, V. J. Muller, B. A.    Petersen, E. L. Isaac, J. Bielicki, B. M. King, C. D. Boulter, A. J.    Moore, N. L. Fazzalari, D. S. Anson, S. Byers, J. J. Hopwood, Enzyme    replacement therapy in a feline model of Maroteaux-Lamy syndrome. J.    Clin. Invest. 97 (1996) 1864-1873.-   [26] E. D. Kakkis, J. Muenzer, G. E. Tiller, L. Waber, J.    Belmont, M. Passage, B. Izykowski, J. Phillips, R. Doroshow, I.    Walot, R. Hoft, E. F. Neufeld, Enzyme-replacement therapy in    mucopolysaccharidosis I. N. Engl. J. Med. 344 (2001) 182-188.-   [27] G. Altarescu, S. Hill, E. Wiggs, N. Jeffries, C. Kreps, C. C.    Parker, R. O. Brady, N. W. Barton, R. Schiffmann, The efficacy of    enzyme replacement therapy in patients with chronic neuronopathic    Gaucher's disease. J. Pediatr. 138 (2001) 539-547.-   [28] C. M. Eng, N. Guffon, W. R. Wilcox, D. P. Germain, P. Lee, S.    Waldek, L. Caplan, G. E. Linthorst, R. J. Desnick, International    Collaborative Fabry Disease Study Group, Safety and efficacy of    recombinant human alpha-galactosidase A-replacement therapy in    Fabry's disease. N. Engl. J. Med. 345 (2001) 9-16.-   [29] F. S. Furbish, C. J. Steer, N. L. Krett, J. A. Barranger,    Uptake and distribution of placental glucocerebrosidase in rat    hepatic cells and effects of sequential deglycosylation. Biochim.    Biophys. Acta. 673 (1981) 425-434.-   [30] G. J. Murray, Lectin-specific targeting of lysosomal enzymes to    reticuloendothelial cells. Methods Enzymol. 149 (1987) 25-42.-   [31] P. D. Stahl, J. S. Rodman, M. J. Miller, P. H. Schlesinger,    Evidence for receptor-mediated binding of glycoproteins,    glycoconjugates, and glycosidases by alveolar macrophages. Proc.    Natl. Acad. Sci. 75 (1978) 1399-1403.-   [32] D. T. Achord, F. E. Brot, C. E. Bell, W. S. Sly, Human    beta-glucuronidase: in vivo clearance and in vitro uptake by a    glycoprotein recognition system on reticuloendothelial cells. Cell    15 (1978) 269-278.-   [33] J. A. Barranger, E. O'Rourke, Lessons learned from the    development of enzyme therapy for Gaucher disease. J. Inherit.    Metab. Dis. 24 (2001) 89-96.-   [34] M. P. Whyte, R. Valdes, L. M. Ryan, W. H. McAlister, Infantile    hypophosphatasia: enzyme replacement therapy by intravenous infusion    of alkaline phosphatase-rich plasma from patients with Paget bone    disease. J. Pediatr. 101 (1982) 379-386.-   [35] M. P. Whyte, W. H. McAlister, L. S. Patton, H. L. Magill, M. D.    Fallon, W. B. Lorentz, H. G. Herrod, Enzyme replacement therapy for    infantile hypophosphatasia attempted by intravenous infusions of    alkaline phosphatase-rich Paget plasma: results in three additional    patients. J. Pediatr. 105 (1984) 926-933.-   [36] M. P. Whyte, H. L. Magill, M. D. Fallon, H. G. Herrod,    Infantile hypophosphatasia: normalization of circulating bone    alkaline phosphatase activity followed by skeletal remineralization.    Evidence for an intact structural gene for tissue nonspecific    alkaline phosphatase. J. Pediatr. 108 (1986) 82-88.-   [37] M. Weninger, R. A. Stinson, H. Plenk, P. Böck, A. Pollak,    Biochemical and morphological effects of human hepatic alkaline    phosphatase in a neonate with hypophosphatasia. Acta Paediatr.    Scand. Suppl. 360 (1989) 154-160.-   [38] M. P. Whyte, M. Landt, L. M. Ryan, R. A. Mulivor, P. S.    Henthorn, K. N. Fedde, J. D. Mahuren, S. P. Coburn, Alkaline    phosphatase: placental and tissue-nonspecific isoenzymes hydrolyze    phosphoethanolamine, inorganic pyrophosphatem and pyridoxal    5′-phosphate. Substrate accumulation in carriers of hypophosphatasia    corrects during pregnancy. J. Clin. Invest. 95 (1995) 1440-1445.-   [39] S, Narisawa, C. Wennberg, J. L. Millan. Abnormal vitamin B6    metabolism in alkaline phosphatase knock-out mice causes multiple    abnormalities, but not the impaired bone mineralization. J. Pathol.    191 (2001) 125-133.-   [40] T. Nishioka, S. Tomatsu, M. A. Gutierrez, K. I. Miyamoto, G. G.    Trandafirescu, P L Lopez, G. H. Grubb, R. Kanai, H. Kobayashi, S.    Yamaguchi, G. S. Gottesman, R. Cahill, A. Noguchi, K.    Miyamoto, W. S. Sly. Enhancement of drug delivery to bone:    Characterization of human tissue-nonspecific alkaline phosphatase    tagged with an acidic oligopeptide. Mol Genet Metab. 2006 July;    88(3):244-255. Epub 2006 Apr. 17.-   [41] Kasugai, S., Fujisawa, R., Waki, Y., Miyamoto, K., and    Ohya, K. (2000) J. Bone. Miner. Res. 15, 936-943-   [42] Yokogawa, K., Miya, K., Sekido, T., Higashi, Y., Nomura, M.,    Fujisawa, R., Morito, K., Masamune, Y., Waki, Y., Kasugai, S., and    Miyamoto, K. (2001) Endocrinology 142, 1228-1233-   [43] Koyama, I., Sakagishi, Y., and Komoda, T. (1986) J. Chromatogr.    374 51-59-   [44] U. K. Laemmli, Cleavage of structural proteins during the    assembly of the head of bacteriophage T4. Nature 227 (1970) 680-685.-   [45] C. R. Merril, D. Goldman, M. L. Van Keuren, Silver staining    methods for polyacrylamide gel electrophoresis. Methods Enzymol.    96 (1983) 230-239.-   [46] S. M. McGEE-RUSSELL, Histochemical methods for calcium. J.    Histochem. Cytochem. 6 (1958) 22-42.-   [47] K. G. Waymire, J. D. Mahuren, J. M. Jaje, T. R. Guilarte, S. P.    Coburn, G. R. MacGregor, Mice lacking tissue non-specific alkaline    phosphatase die from seizures due to defective metabolism of vitamin    B-6. Nat. Genet. 11 (1995) 45-51.-   [48] S, Narisawa, N. Frohlander, J. L. Millan, Inactivation of two    mouse alkaline phosphatase genes and establishment of a model of    infantile hypophosphatasia. Dev. Dyn. 208 (1997) 432-446.

1. A method for increasing life span or increasing bone mineralizationor increasing body weight or reducing epileptic seizure in an animalhaving hypophosphatasia by contacting the animal with an effectiveamount of a composition comprising, a) a secreted soluble anchorlessrecombinant human tissue non-specific alkaline phosphatase (rhTNSALP)encoded by a nucleic acid coding for a rhTNSALP having theglycosylphosphatidylinositol anchoring signal peptide deleted, thenucleic acid expressed by a cell in culture, and the rhTNSALP secretedby the cell, and b) the rhTNSALP being substantially purified, wherebylife span or bone mineralization or body weight is increased in theanimal or epileptic seizure is decreased in the animal.
 2. The method ofclaim 1, the nucleic acid coding for a rhTNSALP consisting of thesequence set forth in amino acid residues 1-505 of SEQ ID NO:1.
 3. Themethod of claim 1, the rhTNSALP having a specific activity of at leastabout 2500 units per milligram of protein.
 4. The method of claim 1,further comprising an injectable aqueous solution.
 5. The method ofclaim 1, wherein the amount of rhTNSALP is equal to a unit dosage. 6.The method of claim 1, the amount of rhTNSALP comprising a unit dosage,the unit dosage being equal to at least about 5 milligrams per kilogramof the animal to be treated.
 7. The method of claim 1, the amount ofrhTNSALP comprising a unit dosage, the unit dosage being equal to atleast about 10 units per gram of the animal to be treated.
 8. A methodfor increasing life span or increasing bone mineralization or increasingbody weight or reducing epileptic seizure in an animal havinghypophosphatasia comprising contacting the animal with an effectiveamount of a composition, the composition including a) a injectableaqueous solution, b) a secreted soluble anchorless recombinant humantissue non-specific alkaline phosphatase (rhTNSALP), i. the rhTNSALPsubstantially purified from the culture media of a cultured cellexpressing a nucleic acid encoding a rhTNSALP having theglycosylphosphatidylinositol anchoring signal peptide deleted, c) theamount of rhTNSALP equal to a unit dosage for the animal. whereby lifespan or bone mineralization or body weight is increased in the animal orepileptic seizure is decreased in the animal.
 9. The method of claim 8,the nucleic acid coding for a rhTNSALP consisting of the sequence setforth in amino acid residues 1-505 of SEQ ID NO:1.
 10. The method ofclaim 8, wherein the unit dosage comprises the amount of rhTNSALP usedto treat the animal for one week.
 11. The method of claim 8, thesubstantially purified rhTNSALP, having a specific activity of at leastabout 2500 units per milligrams of protein.
 12. The method of claim 8,wherein a weekly dosage comprises about 10 units of the rhTNSALP pergram of the animal being treated.
 13. The method of claim 8, wherein aweekly dosage comprises about 5 milligrams of the rhTNSALP per kilogramof the animal to be treated.
 14. The method of claim 8, wherein the unitdosage comprises a single, or a plurality of fractional dosages.
 15. Afor increasing life span or increasing bone mineralization or increasingbody weight or reducing epileptic seizure in an animal havinghypophosphatasia comprising contacting the animal with an effectiveamount of a composition, the composition including a) a injectableaqueous solution, b) a secreted soluble anchorless recombinant humantissue non-specific alkaline phosphatase (rhTNSALP), i. the rhTNSALPsubstantially purified from the culture media of a cultured cellexpressing a nucleic acid encoding a rhTNSALP having theglycosylphosphatidylinositol anchoring signal peptide deleted, and, ii.having a specific activity of at least about 2500 units per milligramsof protein, c) the amount of rhTNSALP equal to a weekly dosage for theanimal, d) the weekly dosage comprising a single, or one or morefractional dosages e) weekly dosage being at least about 10 units of therhTNSALP per gram of the animal to be treated.
 16. The method of claim15, the nucleic acid coding for a rhTNSALP consisting of the sequenceset forth in amino acid residues 1-505 of SEQ ID NO:1.